Topoisomerase-targeting agents

ABSTRACT

The invention provides compounds of formula I or formula II: 
                         
wherein:
 
the bond represented by — is a single bond or a double bond, and R 1 -R 5 , X, and Y have any of the meanings defined in the specification and their pharmaceutically acceptable salts. The invention also provides pharmaceutical compositions comprising a compound of formula I or II, processes for preparing compounds of formula I or II, intermediates useful for preparing compounds of formula I or II, and therapeutic methods for treating cancer and other topoisomerase related conditions using compounds of formula I or II.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/121,292, now U.S. Pat. No. 7,208,492, filed 3 May 2005, whichapplication was a continuation of international application numberPCT/US03/36226, filed 12 Nov. 2003; this application also claimspriority to U.S. Provisional Application No. 60/425,503, filed on 12Nov. 2002 and to U.S. Provisional Application No. 60/425,535 filed on 12Nov. 2002, the specifications of which are herein incorporated byreference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.5RO1CA098127-03 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA-topoisomerases are enzymes which are present in the nuclei of cellswhere they catalyze the breaking and rejoining of DNA strands, whichcontrol the topological state of DNA. Recent studies also suggest thattopoisomerases are also involved in regulating template supercoilingduring RNA transcription. There are two major classes of mammaliantopoisomerases. DNA-topoisomerase-I catalyzes changes in the topologicalstate of duplex DNA by performing transient single-strand breakage-unioncycles. In contrast, mammalian topoisomerase II alters the topology ofDNA by causing a transient enzyme bridged double-strand break, followedby strand passing and resealing. Mammalian topoisomerase II has beenfurther classified as Type II α and Type II β. The antitumor activityassociated with agents that are topoisomerase poisons is associated withtheir ability to stabilize the enzyme-DNA cleavable complex. Thisdrug-induced stabilization of the enzyme-DNA cleavable complexeffectively converts the enzyme into a cellular poison.

Several antitumor agents in clinical use have potent activity asmammalian topoisomerase II poisons. These include adriamycin,actinomycin D, daunomycin, VP-16, and VM-26 (teniposide orepipodophyllotoxin). In contrast to the number of clinical andexperimental drugs which act as topoisomerase II poisons, there arecurrently only a limited number of agents which have been identified astopoisomerase I poisons. Camptothecin and its structurally-relatedanalogs are among the most extensively studied topoisomerase I poisons.Recently, bi- and terbenzimidazoles (Chen et al., Cancer Res. 1993, 53,1332-1335; Sun et al., J. Med. Chem. 1995, 38, 3638-3644; Kim et al., J.Med. Chem. 1996, 39, 992-998), certain benzo[c]phenanthridine andprotoberberine alkaloids and their synthetic analogs (Makhey et al.,Med. Chem. Res. 1995, 5, 1-12; Janin et al., J. Med. Chem. 1975, 18,708-713; Makhey et al., Bioorg. & Med. Chem. 1996, 4, 781-791), as wellas the fungal metabolites, bulgarein (Fujii et al., J. Biol. Chem. 1993,268, 13160-13165) and saintopin (Yamashita et al., Biochemistry 1991,30, 5838-5845) and indolocarbazoles (Yamashita et al., Biochemistry1992, 31, 12069-12075) have been identified as topoisomerase I poisons.Other topoisomerase poisons have been identified including certainbenzo[i]phenanthridine and. cinnoline compounds (see LaVoie et al., U.S.Pat. No. 6,140,328 and WO 01/32631). Despite these reports there iscurrently a need for additional agents that are useful for treatingcancer.

SUMMARY OF THE INVENTION

Applicant has discovered compounds that show inhibitory activity againsttopoisomerase I and/or topoisomerase II, and compounds that areeffective cytotoxic agents against cancer cells, includingdrug-resistant cancer cells. Accordingly, the invention provides acompound of the invention which is a compound of formula I or formulaII:

wherein:

the bond represented by — is a single bond or a double bond;

X is CH or N;

Y is CH or N when the bond represented by — is a double bond, or Y isCH₂ or NR_(x) when the bond represented by — is a single bond;

one of R₁ and R₂ is nitro, halo, or (C₁-C₆)alkoxy and the other ishydrogen, nitro, halo, or (C₁-C₆)alkoxy; or R₁ and R₂ taken together aremethylenedioxy;

one of R₃ and R₄is nitro, halo, or (C₁-C₆)alkoxy and the other of R₃ andR₄is hydrogen, nitro, halo, or (C₁-C₆)alkoxy; or R₃ and R₄ takentogether are methylenedioxy;

R₅ is a solubilizing group; and

R_(x) is hydrogen or (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof. Preferably, Y is not N,when R₅is —OH.

The invention also provides a pharmaceutical composition comprising aeffective amount of a compound of the invention in combination with apharmaceutically acceptable diluent or carrier.

The invention also provides a method of inhibiting cancer cell growth,comprising administering to a mammal afflicted with cancer, an amount ofa compound of the invention, effective to inhibit the growth of saidcancer cells.

The invention also provides a method comprising inhibiting cancer cellgrowth by contacting said cancer cell in vitro or in vivo with an amountof a compound of the invention, effective to inhibit the growth of saidcancer cell.

The invention also provides a method for modulating topoisomeraseactivity in a mammal in need of such treatment comprising administeringto the mammal, an amount of a compound of the invention effective toprovide a topoisomerase modulating effect.

The invention also provides a compound of the invention for use inmedical therapy, preferably for use in treating cancer, for example,solid tumors, as well as the use of a compound of the invention for themanufacture of a medicament useful for the treatment of cancer, forexample, solid tumors.

The invention also provides the use of a compound of the invention forthe manufacture of a medicament useful for modulating the activity of atopoisomerase.

The invention also provides processes and novel intermediates disclosedherein (e.g. in the Schemes hereinbelow) which are useful for preparingcompounds of the invention. Some of the compounds of formula I and IIare useful to prepare other compounds of formula I and II.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described.

-   -   “(C₁-C₆)alkyl” denotes a straight or branched carbon chain with        1, 2, 3, 4, 5, or 6, carbon atoms, but reference to an        individual radical such as “propyl” embraces only the straight        chain radical, a branched chain isomer such as “isopropyl” being        specifically referred to.    -   “(C₃-C₆)cycloalkyl” denotes a carbocyclic ring with 3, 4, 5, or        6, carbon atoms.    -   “Aryl” denotes a phenyl radical or an ortho-fused bicyclic        carbocyclic radical having about nine to ten ring atoms in which        at least one ring is aromatic. Examples of aryl include phenyl,        indenyl, and naphthyl.    -   “Aryl(C₁-C₆)alkyl” refers to a group of the formula        aryl-(C₁-C₆)alkyl-, where aryl and (C₁-C₆)alkyl are as defined        herein.    -   “Halo” is fluoro, chloro, bromo, or iodo.    -   “Solubilizing group” is a substituent that increases the water        solubility of the compound of formula I or II compared to the        corresponding compound lacking the substituent (i.e. wherein the        substituent is hydrogen). Examples of solubilizing groups        include (C₁-C₆)alkoxycarbonyl (e.g. —CO₂Me), cyano, halo,        hydroxy, mercapto, oxo (═O), carboxy (COOH), nitro,        pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,        piperazinyl, morpholinyl, thiomorpholinyl, and —NR_(f)R_(g),        wherein R_(f) and R_(g) may be the same or different and are        chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

A specific value for X is N.

A specific value for X is CH.

A specific value for Y is N.

A specific value for Y is CH.

A specific value for R₁ is nitro.

A specific value for R₁ is hydrogen.

A specific value for R₁ is halo.

A specific value for R₁ is (C₁-C₆)alkoxy.

A specific value for R₂ is nitro.

A specific value for R₂ is hydrogen.

A specific value for R₂ is halo

A specific value for R₂ is (C₁-C₆)alkoxy.

A specific value for R₁ and R₂ taken together is methylenedioxy.

Specifically R₃ and R₄ are both nitro.

Specifically R₃ and R₄ are both halo.

Specifically R₃ and R₄ are both chloro.

Specifically R₃ and R₄ are both (C₁-C₆)alkoxy (e.g. methoxy).

Specifically one of R₃ and R₄ is hydrogen and the other is nitro.

Specifically one of R₃ and R₄ is hydrogen and the other is(C₁-C₆)alkoxy.

Specifically one of R₃ and R₄ is hydrogen and the other is halo.

Specifically one of R₃ and R₄ is hydrogen and the other is chloro.

A specific value for R₃ and R₄ taken together is methylenedioxy.

Specifically R₁ and R₂ taken together are methylenedioxy; and R₃ and R₄are each methoxy.

Specifically R₅ is (C₁-C₆)alkoxycarbonyl, cyano, halo, hydroxy,mercapto, carboxy, nitro, pyrrolidinyl, piperidinyl, imidazolidinyl,imidazolinyl, piperazinyl, morpholinyl, thiomorpholinyl, or—NR_(f)R_(g), wherein R_(f) and R_(g) may be the same or different andare chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically R₅ is (C₁-C₆)alkoxycarbonyl, cyano, carboxy, or—NR_(f)R_(g), wherein R_(f) and R_(g) may be the same or different andare chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically R₅ is —W-Z wherein: W is absent, —(C═O)—, —CH₂—, —(C═O)O—,—O(C═O)—, —O—, —NR_(a)(C═O)—, —C(═O)NR_(a)—, or —NR_(a)—; Z is(C₁-C₄)alkyl substituted with one or more water solubilizing groups; andR_(a) is hydrogen or (C₁-C₄)alkyl.

A specific value for W is absent.

A specific value for W is —(C═O)—.

A specific value for W is —CH₂—.

A specific value for W is —(C═O)O—.

A specific value for W is —O(C═O)—.

A specific value for W is —O—.

A specific value for W is —NR_(a)(C═O)—.

A specific value for W is —C(═O)NR_(a)—.

A specific value for W is —NR_(a)—.

A specific value for Z is (C₁-C₄)alkyl substituted with one or two watersolubilizing groups.

A specific value for Z is (C₁-C₄)alkyl substituted with one watersolubilizing group.

A specific value for W is —O—.

A specific value for W is —NH—.

A specific value for Z is —CH₂—R_(a) where R_(a) is (C₁-C₃)alkylsubstituted with one or two water solubilizing groups.

A specific value for Z is —CH₂—R_(a) where R_(a) is (C₁-C₃)alkylsubstituted with one water solubilizing groups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more hydroxygroups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one hydroxy group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more mercaptogroups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one mercapto group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more carboxygroups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one carboxy group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or moreNR_(f)R_(g) groups; wherein R_(f) and R_(g) may be the same or differentand are chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one NR_(f)R_(g)group; wherein R_(f) and R_(g) may be the same or different and arechosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more NH₂groups.

Specifically —W-Z is a (C₁-C₄)alkyl substituted with one NH₂ group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more N(CH₃)₂groups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one N(CH₃)₂ group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or moreN(CH₂CH₃)₂ groups.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one N(CH₂CH₃)₂ group.

Specifically —W-Z is (C₁-C₄)alkyl substituted with one or more(C₁-C₆)alkoxycarbonyl , cyano, halo, hydroxy, mercapto, oxo, carboxy,nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,piperazinyl, morpholinyl, thiomorpholinyl, or —NR_(f)R_(g) groups,wherein R_(f) and R_(g) may be the same or different and are chosen fromhydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄)alkyl substituted with one or two groupsselected from hydroxy, mercapto, carboxy, amino, methylamino,ethylamino, dimethylamino, and diethylamino.

Specifically —W-Z is 2-hydroxyethyl.

Specifically —W-Z is 3-hydroxypropyl.

Specifically —W-Z is 2-hydroxypropyl.

Specifically —W-Z is —CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently hydrogen or (C₁-C₆)alkyl.

Specifically —W-Z is —CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently methyl or ethyl.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or more hydroxygroups.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one hydroxy group.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or more mercaptogroups.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one mercapto group.

Specifically —W-Z is (C₁-C₄)alkoxy substituted with one or more carboxygroups.

Specifically —W-Z is (C₁-C₄)alkoxy substituted with one carboxy group.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or moreNR_(f)R_(g) groups; wherein R_(f) and R_(g) may be the same or differentand are chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one NR_(f)R_(g)group; wherein R_(f) and R_(g) may be the same or different and arechosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or more NH₂groups.

Specifically —W-Z is a (C₂-C₄)alkoxy substituted with one NH₂ group.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or more N(CH₃)₂groups.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one N(CH₃)₂ group.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or moreN(CH₂CH₃)₂ groups.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one N(CH₂CH₃)₂group.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or more(C₁-C₆)alkoxycarbonyl , cyano, halo, hydroxy, mercapto, oxo, carboxy,nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,piperazinyl, morpholinyl, thiomorpholinyl, or —NR_(f)R_(g) groups,wherein R_(f) and R_(g) may be the same or different and are chosen fromhydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄)alkoxy substituted with one or two groupsselected from hydroxy, mercapto, carboxy, amino, methylamino,ethylamino, dimethylamino, and diethylamino.

Specifically —W-Z is 2-hydroxyethoxy.

Specifically —W-Z is 3-hydroxypropoxy.

Specifically —W-Z is 2-hydroxypropoxy.

Specifically —W-Z is —OCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently hydrogen or (C₁-C₆)alkyl.

Specifically —W-Z is —OCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently methyl or ethyl.

Specifically —W-Z is (C₂-C₄)alkylamino substituted with one or morehydroxy groups.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one hydroxygroup.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or moremercapto groups.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one mercaptogroup.

Specifically —W-Z is (C₁-C₄) alkylamino substituted with one or morecarboxy groups.

Specifically —W-Z is (C₁-C₄) alkylamino substituted with one carboxygroup.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or moreNR_(f)R_(g) groups; wherein R_(f) and R_(g) may be the same or differentand are chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one NR_(f)R_(g)group; wherein R_(f) and R_(g) may be the same or different and arechosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or more NH₂groups.

Specifically —W-Z is a (C₂-C₄) alkylamino substituted with one NH₂group.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or moreN(CH₃)₂ groups.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one N(CH₃)₂group.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or moreN(CH₂CH₃)₂ groups.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one N(CH₂CH₃)₂group.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or more(C₁-C₆)alkoxycarbonyl , cyano, halo, hydroxy, mercapto, oxo, carboxy,nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,piperazinyl, morpholinyl, thiomorpholinyl, or —NR_(f)R_(g) groups,wherein R_(f) and R_(g) may be the same or different and are chosen fromhydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄) alkylamino substituted with one or twogroups selected from hydroxy, mercapto, carboxy, amino, methylamino,ethylamino, dimethylamino, and diethylamino.

Specifically —W-Z is 2-hydroxyethylamino.

Specifically —W-Z is 3-hydroxypropylamino.

Specifically —W-Z is 2-hydroxypropylamino.

Specifically —W-Z is —NHCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently hydrogen or (C₁-C₆)alkyl.

Specifically —W-Z is —NHCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently methyl or ethyl.

Specifically —W-Z is (C₁-C4)alkanoyl substituted with one or morehydroxy groups.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one hydroxy group.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or moremercapto groups.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one mercaptogroup.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or morecarboxy groups.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one carboxy group.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or moreNR_(f)R_(g) groups; wherein R_(f) and R_(g) may be the same or differentand are chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one NR_(f)R_(g)group; wherein R_(f) and R_(g) may be the same or different and arechosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or more NH₂groups.

Specifically —W-Z is a (C₁-C₄)alkanoyl substituted with one NH₂ group.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or moreN(CH₃)₂ groups.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one N(CH₃)₂ group.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or moreN(CH₂CH₃)₂ groups.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one N(CH₂CH₃)₂group.

Specifically —W-Z is (C₁-C₄)alkanoyl substituted with one or more(C₁-C₆)alkoxycarbonyl, cyano, halo, hydroxy, mercapto, oxo, carboxy,nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,piperazinyl, morpholinyl, thiomorpholinyl, or —NR_(f)R_(g) groups,wherein R_(f) and R_(g) may be the same or different and are chosen fromhydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₂-C₄)alkanloyl substituted with one or two groupsselected from hydroxy, mercapto, carboxy, amino, methylamino,ethylamino, dimethylamino, and diethylamino.

Specifically —W-Z is 2-hydroxyethanoyl.

Specifically —W-Z is 3-hydroxypropanoyl.

Specifically —W-Z is 2-hydroxypropanoyl.

Specifically —W-Z is —C(O)CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently hydrogen or (C₁-C₆)alkyl.

Specifically —W-Z is —C(O)CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f) areeach independently methyl or ethyl.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore hydroxy groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with onehydroxy group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore mercapto groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with onemercapto group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore carboxy groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with onecarboxy group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore NR_(f)R_(g) groups; wherein R_(f) and R_(g) may be the same ordifferent and are chosen from hydrogen, (C₁-C₆)alkyl, and(C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with oneNR_(f)R_(g) group; wherein R_(f) and R_(g) may be the same or differentand are chosen from hydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore NH₂ groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one NH₂group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore N(CH₃)₂ groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with oneN(CH₃)₂ group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore N(CH₂CH₃)₂ groups.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with oneN(CH₂CH₃)₂ group.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ormore (C₁-C₆)alkoxycarbonyl, cyano, halo, hydroxy, mercapto, oxo,carboxy, nitro, pyrrolidinyl, piperidinyl, imidazolidinyl, imidazolinyl,piperazinyl, morpholinyl, thiomorpholinyl, or —NR_(f)R_(g) groups,wherein R_(f) and R_(g) may be the same or different and are chosen fromhydrogen, (C₁-C₆)alkyl, and (C₃-C₆)cycloalkyl.

Specifically —W-Z is (C₁-C₄)alkylaminocarbonyl substituted with one ortwo groups selected from hydroxy, mercapto, carboxy, amino, methylamino,ethylamino, dimethylamino, and diethylamino.

Specifically —W-Z is 2-hydroxyethylaminocarbonyl.

Specifically —W-Z is 3-hydroxypropylaminocarbonyl.

Specifically —W-Z is 2-hydroxypropylaminocarbonyl.

Specifically —W-Z is —C(O)NHCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f)are each independently hydrogen or (C₁-C₆)alkyl.

Specifically —W-Z is —C(O)NHCH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f)are each independently methyl or ethyl.

Specifically —W-Z is —NHC(O)CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f)are each independently hydrogen or (C —C₆)alkyl.

Specifically —W-Z is —NHC(O)CH₂CH₂—NR_(f)R_(g) wherein R_(f) and R_(f)are each independently methyl or ethyl.

A specific compound is a compound of formula I or II wherein R₁ and R₂taken together are methylenedioxy; and R₃ and R₄ are each methoxy; or apharmaceutically acceptable salt thereof.

A specific compound of the invention is a compound of formula I or IIwherein the bond represented by — is a single bond.

A specific compound of the invention is a compound of formula I or IIwherein the bond represented by — is a double bond.

A specific compound of the invention is a compound of formula I orformula II:

wherein:

the bond represented by — is a single bond or a double bond;

X is CH or N;

Y is CH or N when the bond represented by — is a double bond, or Y isCH₂ or NR_(a) when the bond represented by — is a double bond;

one of R₁ and R₂ is nitro and the other is hydrogen, halo, or nitro; orR₁ and R₂ taken together are methylenedioxy;

one of R₃ and R₄ is nitro, halo, or (C₁-C₆)alkoxy and the other of R₃and R₄ is hydrogen, nitro, halo, or (C₁-C₆)alkoxy;

R₅ is a solubilizing group; and

R_(a) is hydrogen or (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof;

provided that Y is not N, when R₅ is —OH.

Another specific compound of the invention is a compound of thefollowing formula:

wherein:

X and Y are each independently CH or N;

one of R₁ and R₂ is nitro and the other is hydrogen, or nitro; or R₁ andR₂ taken together are methylenedioxy;

one of R₃ and R₄ is nitro, halo, or (C₁-C₆)alkoxy and the other of R₃and R₄ is hydrogen, nitro, halo, or (C₁-C₆)alkoxy; and

R₅ is a solubilizing group;

or a pharmaceutically acceptable salt thereof.

Another specific compound of the invention is a compound of thefollowing formula:

wherein:

X and Y are each independently CH or N;

one of R₁ and R₂ is nitro and the other is hydrogen, or nitro; or R₁ andR₂ taken together are methylenedioxy;

one of R₃ and R₄ is nitro, halo, or (C₁-C₆)alkoxy and the other of R₃and R₄ is hydrogen, nitro, halo, or (C₁-C₆)alkoxy; and

R₅ is a solubilizing group;

or a pharmaceutically acceptable salt thereof.

A specific compound of the invention is a compound of formula III:

or a pharmaceutically acceptable salt thereof; wherein R₁-R₅, X and Yhave any of the values or specific values defined herein.

A specific compound of the invention is a compound of IV:

or a pharmaceutically acceptable salt thereof; wherein R₁-R₅, X and Yhave any of the values or specific values defined herein.

A specific compound of the invention is a compound of formula V:

or a pharmaceutically acceptable salt thereof; wherein R₁-R₅, X and Yhave any of the values or specific values defined herein.

A specific compound of the invention is a compound of formula VI.

or a pharmaceutically acceptable salt thereof; wherein R₁-R₅, X and Yhave any of the values or specific values defined herein.

For a compound of formula I, II, III, and V when the bond represented by— is a single bond, the additional valence on the carbon bearing R₅ thatis not shown in the formula is occupied by a hydrogen.

A compound of formula I can be prepared, for example, as described inSchemes 1-4 below.

Typically, preferred compounds of the invention demonstrate someselectivity for Topoisomerase I compared to Topoisomerase II.

Representative compounds of formula I wherein: X is CH or N; Y is CH; Wis C(═)O, C(═)OO; or CH₂; and Z is N(CH₃)₂, CH₂N(CH₃)₂, or CH₂CH₂N(CH₃)₂can be prepared as illustrated in Scheme 1. Compounds 4, 5, 6, 9, 10,11, 12 and 13 are representative compounds of formula 1.

Condensation of the benzaldehyde 1 with the 4-quinolylacetonitrile 2will provide 3, which in a palladium-catalyzed cyclization (orphoto-initiated cyclization) will provide the cyano intermediate 4.Reduction of 4 will provide the benzylamine, 5, which can be convertedto its N,N-dimethyl derivative, 6. The carboxylic acid 9 can be preparedby hydrolysis of 4. Alternatively, reaction of 1 and 7 will form 8,which upon palladium-catalyzed cyclization (or photo-initiatedcyclization) followed by hydrolysis will form 9. Tin coupling performedby reacting N,N-dialkylaminomethyltributyl tin with the acid chloride of9 will provide the α-dimethylaminoketone 10, which can be reduced to the2-(N,N-dimethylaminoethyl) derivative, 11. Alternatively, the acidchloride of 9 can be reacted with various nucleophiles, such asN,N-dimethylethanolamine or N,N-dimethylethylenediamine to form theester, 12, or amide, 13, respectively.

Representative compounds of formula I wherein X is CH or N; Y is CH; Wis O or NH; and Z is CH₂CH₂N(CH₃)₂ can be prepared as illustrated inScheme 2. Compounds 18, 19, 20, 21, 22, and 23 are representativecompounds of formula I.

Reaction of the anion of the dithiane 15 with the benzyl bromide 14 willprovide 16, which can be converted to the ketone 17. Palladium-catalyzedcyclization (or photo-initiated cyclization) of 17 will provide 18 whichcan be treated with a variety of electrophiles, includingN,N-dimethyl-2-chloroethanamine which would provide 19. In addition, 18could be acylated to provide the ester 20. Alternatively, 18 could beconverted to the aryl amine 21, which could be similarly alkylated oracylated to provide 22 and 23, respectively.

Representative compounds of formula I wherein X is either CH or N; Y isN; W is CO, COO; or CH₂; and Z is N(CH₃)₂; CH₂N(CH₃)₂; CH₂CH₂N(CH₃)₂ canbe prepared as illustrated in Scheme 3. Compounds 29, 30, 31, 32, 33,and 34 are representative compounds of formula I.

Reaction of 24 with the acid chloride of 25 will provide the amide 26,which in the presence of reagents such as phosphorous oxychloride willprovide the chloro intermediate 27. Reaction of 27 with cyanide anionwill provide 28, which can undergo palladium-catalyzed cyclization (orphoto-initiated cyclization) and hydrolysis to give the acid 29.Treatment of the acid chloride of 29 with N,N-dialkylaminomethyltributyltin will provide the α-aminoketone 31. Treatment of the acid chloride of29 with 2-(N,N-dimethylamino)ethanol or N,N-dimethylethylenediamine willprovide 30 and 32, respectively. Palladium-catalyzed cyclization (orphoto-initiated cyclization) of 28 will provide 33. Reduction of 33followed by methylation of the resulting amine will provide 34.

Representative compounds of formula I wherein X is CH or N; Y is N; W isO or NH; and Z is CH₂CH₂N(CH₃)₂ can be prepared as outlined-in Scheme 4.Compounds 36, 39 and 40 are representative compounds of formula 1.

Scheme 4 illustrates three methods outlined for the preparation ofcompound 39. One involves direct alkylation of the enol form of compound35. The second involves displacement of the chloro substituent onintermediate 36 with the alkoxide of 2-(N,N-dimethylamino)ethanol. Thethird alternative is to react 27 with 2-(N,N-dimethylamino)ethanol andto subject the resulting ether, 37, to palladium-catalyzed cyclizationconditions (or photo-initiated cyclization). The two approaches to form40 are similar. In one approach N,N-dimethylethylenediamine is reactedwith 36. In the other N,N-dimethylethylenediamine is first reacted with27 and the resulting product, 38, is subject to palladium-catalyzedcyclization conditions (or photo-initiated cyclization).

Representative compounds of formula II wherein: X is CH or N; Y is CH; Wis CO, COO, or CH₂; and Z is N(CH₃)₂, CH₂N(CH₃)₂, or CH₂CH₂N(CH₃)₂ canbe prepared as illustrated in Scheme 5. Compounds 104, 105, 106, 109,110, 111, 112 and 113 are representative compounds of formula II.

Condensation of the phenylacetonitrile 101 with 6,7-substituted4-formylquinoline 102 will yield 103, which in a palladium-catalyzedcyclization (or photo-initiated cyclization) can be converted to thecyano intermediate 104. Reduction of 104 will provide the benzylamine,105, which can be converted to its N,N-dimethyl derivative, 106. Thecarboxylic acid 109 can be prepared by hydrolysis of 104. Alternatively,reaction of 100 and 107 will form 108, which upon palladium-catalyzedcyclization (or photo-initiated cyclization) followed by hydrolysis willform 109. Tin coupling performed by reactingN,N-dialkylaminomethyltributyl tin with the acid chloride of 109 willprovide the α-dimethylaminoketone 110, which can be reduced to the2-(N,N-dimethylaminoethyl) derivative, 111. Alternatively, the acidchloride of 109 can be reacted with various nucleophiles, such asN,N-dimethylethanolamine or N,N-dimethylethylenediamine to form theester, 112, or amide, 113, respectively.

Representative compounds of formula II wherein: X is CH or N; Y is CH; Wis O or NH; and Z is CH₂CH₂N(CH₃)₂ can be prepared as outlined in Scheme6. Compounds 118, 119, 120, 121, 122, and 123 are representativecompounds of formula II.

Reaction of the anion of the dithiane 114 with the benzyl bromide 115will provide 116, which can be converted to the ketone 117.Palladium-catalyzed cyclization of 117 (or photo-initiated cyclization)will provide 118 which can be treated with a variety of electrophiles,including N,N-dimethyl-2-chloroethanamine which would provide 119. Inaddition, it could be acylated to form 120. Alternatively, 118 could beconverted to the aryl amine 121, which could be similarly alkylated oracylated to provide 122 and 123, respectively.

Representative compounds of formula II wherein: X is CH or N; Y is N; Wis CO, COO, or CH₂; and Z is N(CH₃)₂, CH₂N(CH₃)₂, or CH₂CH₂N(CH₃)₂ canbe prepared as outlined in Scheme 7. Compounds 129, 130, 131, 133, 132,134 are representative compounds of formula II.

Reaction of 125 with the acid chloride of 124 will provide the amide126, which in the presence of reagents such as phosphorous oxychloridewill provide the chloro intermediate 27. Reaction of 127 with cyanideanion will provide 128, which can undergo palladium-catalyzedcyclization (or photo-initiated cyclization) and hydrolysis to give theacid 129. Treatment of the acid chloride of 129 withN,N-dialkylaminomethyltributyl tin will provide the α-aminoketone 131.Treatment of the acid chloride of 129 with 2-(N,N-dimethylamino)ethanolor N,N-dimethylaminethylenediamine will provide 130 and 132,respectively. Palladium-catalyzed cyclization of 128 (or photo-initiatedcyclization) will provide 133. Reduction of 133 followed by methylationof the resulting amine will provide 134.

Representative compounds of formula II wherein: X is CH or N; Y is N; Wis O or NH; and Z is CH₂CH₂N(CH₃)₂ can be prepared as illustrated inScheme 8. Compounds 136, 139 and 140 are representative compounds offormula II.

There are three methods outlined for the preparation of compound 139.One involves direct O-alkylation of compound 135. The second involvesdisplacement of the chloro substituent on intermediate 136 with thealkoxide of 2-(N,N-dimethylamino)ethanol. The third alternative is toreact 127 with 2-(N,N-dimethylamino)ethanol and to subject the resultingether, 137, to palladium-catalyzed cyclization conditions (orphoto-initiated cyclization). The two approaches to form 140 aresimilar. In one approach N,N-dimethylethylenediamine is reacted with136. In the other N,N-dimethylethylenediamine is first reacted with 127and the resulting product, 138, is subjected to palladium-catalyzedcyclization conditions (or photo-initiated cyclization).

Representative compounds of formula II wherein: X is CH or N; Y is CH₂;and the bond represented by — is a single bond can be prepared asillustrated in Scheme 9.

Reduction of ester 141 provides the alcohol 142. Palladium-catalyzedcyclization (or photo-initiated cyclization) of compound 142 providesthe compound of the invention 143. Compound 143 is also a usefulintermediate that can be used to prepare a variety of other compounds ofthe invention.

Representative compounds of formula I wherein: X is CH or N; Y is CH₂;and the bond represented by — is a single bond can be prepared asillustrated in Scheme 10.

Reduction of ester 144 provides the alcohol 145. Palladium-catalyzedcyclization (or photo-initiated cyclization) of compound 145 providesthe compound of the invention 146. Compound 146 is also a usefulintermediate that can be used to prepare a variety of other compounds ofthe invention.

The starting materials employed in the synthetic methods describedherein are commercially available, have been reported in the scientificliterature, or can be prepared from readily available starting materialsusing procedures known in the field. It may be desirable to optionallyuse a protecting group during all or portions of the above describedsynthetic procedures. Such protecting groups and methods for theirintroduction and removal are well known in the art. See Greene, T. W.;Wutz, P. G. M. “Protecting Groups In Organic Synthesis” second edition,1991, New York, John Wiley & Sons, Inc.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine topoisomerase inhibition activityor cytotoxic activity using the standard tests described herein, orusing other similar tests which are well known in the art.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal, for example, sodium,potassium or lithium, or alkaline earth metal, for example calcium,salts of carboxylic acids can also be made.

The compounds of formula I and II can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, that is, orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, forexample, orally, in combination with a pharmaceutically acceptablevehicle such as an inert diluent or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of the invention to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the invention can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of the invention in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg per day, e.g., from about 1 to about 60 mg/kg ofbody weight per day or about 2 to 50 mg/kg per day.

The compound may conveniently be administered in unit dosage form; forexample, containing 5 to 1,000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The ability of a compound of the invention to effect topoisomerase I orII mediated DNA cleavage can be determined using pharmacological modelsthat are well known to the art, for example, using a model like Test Adescribed below.

Test A. Topoisomerase-mediated DNA Cleavage Assays.

Human topoisomerase I was expressed in E. Coli and isolated as arecombinant fusion protein using a T7 expression system as describedpreviously (Gatto, B., Sanders, M. M., Yu, C., Wu, H.-Y., Makhey, D.,LaVoie, E. J., and Liu, L. F. (1996) Cancer Res. 56, 2795-2800).Recombinant human topoisomerase IIα was isolated and purified aspreviously described (Wasserman, R. A. Austin, C. A., Fisher, L. M.;Wang, J. C., Cancer Res., 1993, 53, 3591; Halligan, B. D.; Edwards, K.A.; Liu, L. F. J. Biol. Chem. 1985, 260, 2475). Plasmid YepG was alsopurified by the alkali lysis method followed by phenol deproteinationand CsCl/ethidium isopycnic centrifugation method as described. Theend-labeling of the plasmid was accomplished by digestion with arestriction enzyme followed by end-filling with Klenow polymerase aspreviously described (Maniatis, T.; Fritsch, E. F.; Sambrook, J.Molecular Cloning, a Laboratory Manual; Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. 1982; pp 149-185.). The cleavage assays wereperformed as previously reported (Gatto, B., Sanders, M. M., Yu, C., Wu,H.-Y., Makhey, D., LaVoie, E. J., and Liu, L. F. (1996) Cancer Res. 56,2795-2800; Tewey, K. M., Rowe, T. C., Yang, L., Hallogan, B. C., andLiu, L. F. (1984) Science 226, 466-468; Li T-K., Chen A Y, Yu C, Mao Y,Wang H, Liu L F. (1999) Genes Dev 13(12):1553-60; Wang, H.; Mao, Y.;Chen, A. Y.; Zhou, N.; and LaVoie, E. J.; Liu, L. F. Biochemistry, 2001,40, 3316). The drug and the DNA in presence of topoisomerase I wasincubated for 30 minutes at 37° C. After development of the gels,typically 24-hour exposure was used to obtain autoradiograms outliningthe extent of DNA fragmentation. Topoisomerase I-mediated DNA cleavagevalues are reported as REC, Relative Effective Concentration, i.e.concentrations relative to topotecan, whose value is arbitrarily assumedas 1.0, that are able to produce the same cleavage on the plasmid DNA inthe presence of human topoisomerase I. Topoisomerase II-mediated DNAcleavage values are reported as REC, Relative Effective Concentration,potency was based upon the relative amount of drug needed to induceapproximately 10% DNA fragmentation, i.e. concentrations relative toVM-26, whose value is arbitrarily assumed as 1.0, that are able toproduce the same cleavage on the plasmid DNA in the presence of humantopoisomerase II. The relative effective concentration (REC) values ascompared to camptothecin that results in approximately 10% cleavage ofDNA for representative compounds of the invention are typically in therange of from about 0.1 to about 100.

The cytotoxic effects of a compound of the invention can be determinedusing pharmacological models that are well known to the art, forexample, using a model like Test B described below.

Test B. Inhibition of Cell Growth: MTT-microtiter Plate TetrazoliniumCytotoxicity Assay (RPMI 8402, CPT-K5, U937, U937/CR Cells)

The cytotoxicity is determined using the MTT-microtiter platetetrazolinium cytotoxicity assay (MTA), see Chen A. Y. et al. CancerRes. 1993, 53, 1332; Mosmann, T. J., J. Immunol. Methods 1983, 65, 55;and Carmichael, J. et al. Cancer Res. 1987, 47, 936. The humanlymphoblast RPMI 8402 and its camptothecin-resistant variant cell line,CPT-K5 were provided by Dr. Toshiwo Andoh (Anchi Cancer ResearchInstitute, Nagoya, Japan), see Andoh, T.; Okada, K, Adv. in Pharmacology1994, 29B, 93. Human U-937 myeloid leukemia cells and U-937/CR cellswere described by Rubin et al., J. Biol. Chem., 1994, 269, 2433-2439.The cytotoxicity assay is performed by using 96-well microtiter platesusing 2000 cells/well, in 200 mL of growth medium. Cells are grown insuspension at 37° C. in 5% CO₂ and maintained by regular passage in RPMImedium supplemented with 10% heat-inactivated fetal bovine serum,L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (0.1 mg/mL).For determination of IC₅₀, cells are exposed continuously for 3-4 daysto varying concentrations of drug, and MTT assays were performed at theend of the fourth day. Each assay is performed with a control that didnot contain any drug. All assays are performed at least twice in 6replicate wells. All assays are performed under the direction of Dr. L.F. Liu, Department of Pharmacology, The University of Medicine andDentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway,N.J. Representative compounds of the invention typically demonstrate anIC₅₀ of less than 1 micromolar in this assay.

The compounds of the invention can function as cytotoxic agents againsttumor cell lines, including multi-drug resistant tumor cell lines. Thus,the compounds are useful to treat cancer and can be used to treat tumorsthat are resistant to other specific chemotherapeutic agents.

Topoisomerase inhibitors are also known to possess antibacterial,antifungal, antiprotozoal, antihelmetic, and antiviral activity.Accordingly, the topoisomerase inhibitors of the invention may also beuseful as antibacterial, antifungal, antipsoritic (psoriasis)antiprotozoal, antihelmetic, or antiviral agents. In particular,compounds of the invention that demonstrate little or no activity asmammalian topoisomerase I poisons, because of the possibility of similarmolecular mechanism of action, could be highly active and selectiveantibacterial, antifungal, antiprotozoal, antihelmetic, or antiviralagents. Thus, certain compounds of the invention may be particularlyuseful as systemic antibacterial, antifungal, antiprotozoal,antihelmetic, or antiviral agents in mammals. The invention alsoprovides the use of a compound of the invention for the manufacture of amedicament useful for producing an antibacterial, antifungal,antiprotozoal, antihelmetic, or antiviral effect in a mammal.

As used herein, the term “solid mammalian tumors” include cancers of thehead and neck, lung, mesothelioma, mediastinum, esophagus, stomach,pancreas, hepatobiliary system, small intestine, colon, rectum, anus,kidney, ureter, bladder, prostate, urethra, penis, testis, gynecologicalorgans, ovarian, breast, endocrine system, skin central nervous system;sarcomas of the soft tissue and bone; and melanoma of cutaneous andintraocular origin. The term “hematological malignancies” includeschildhood leukemia and lymphomas, Hodgkin's disease, lymphomas oflymphocytic and cutaneous origin, acute and chronic leukemia, plasmacell neoplasm and cancers associated with AIDS. The preferred mammalianspecies for treatment are humans and domesticated animals.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES

Representative compounds of the invention were prepared as illustratedbelow in Schemes A-C.

Example 1 Synthesis of Compound 205

A solution of compound 204 (450 mg, 0.85 mmol) in acetonitrile (800 mg)was transferred to the photoreactor apparatus and was degassed bynitrogen purge for 30 minutes. The solution was then irradiated througha vycor filter for 45 minutes. The mixture was removed from thephotoreactor, and an equal portion of compound 204 (450 mg, 0.85 mmol)in acetonitrile (800 mg) was reacted according to the same procedure.The cyclized product, which had precipitated out during the course ofthe reaction, was isolated by filtration and was washed with additionalacetonitrile. Thorough drying yielding 348 mg, in 51% yield; ¹H NMR(CDCl₃+1 drop CD₃OD) δ 1.48 (t, 3H, J=7.1), 3.98 (s, 3H), 4.13 (s, 3H),4.53 (q, 2H, J=7.1), 6.26 (s, 2H), 7.73 (s, 1H), 7.93 (s, 1H), 8.11 (s,1H), 8.18 (s, 1H), 8.70 (s, 1H), 10.24 (s, 1H); ¹³C NMR (CDCl₃+1 dropCD₃OD) δ 14.2, 55.9, 56.6, 62.5, 99.5, 100.0, 102.9, 103.8, 106.6,120.4, 121.2, 123.2, 124.7, 126.8, 131.8, 132.7, 135.0, 139.8, 151.4,151.5, 152.3, 152.4, 166.7; HRMS calcd for C₂₃H₁₉NO₆H: 406.1290; found406.1270.

The intermediate compound 204 was prepared as follows.

a. Synthesis of compound 201. Iron (III) chloride (54.2 g, 0.33 mol) wasdissolved in glacial acetic acid (600 mL) with warming to 60° C.3,4-Methylenedioxyaniline (27.4 g, 0.2 mol) was added and the mixturewas stirred for 5 minutes. Methyl vinyl ketone (17.4 mL, 0.21 mol) wasadded dropwise over five minutes. Following the completion of theaddition, the mixture was heated to reflux with stirring for 1.5 hours.The mixture was cooled and the precipitate was filtered and washed withadditional acetic acid. This material was then neutralized by additionto cold 30% NaOH, and the resulting mixture was filtered and air-dried.The crude material was then extracted with chloroform (7×200 mL), andthe combined extracts were washed with 10% K₂CO₃ (3×300 mL) and weredried (MgSO₄) and concentrated under vacuum. The resulting material wasrecrystallized from ethyl ether, yielding 16.6 g as a fluffy light beigesolid, in 44% yield; mp 100.5-101.5° C.; ¹H NMR (CDCl₃) δ 2.51 (s, 3H),6.04 (s, 2H), 7.02 (d, 1H, J=4.4), 7.13 (s, 1H), 7.32 (s, 1H), 8.52 (d,1H, J=4.4); ¹³C NMR (CDCl₃) δ 19.1, 99.3, 101.7, 106.3, 120.6, 125.0,142.9, 146.3, 147.8, 147.9, 150.2; HRMS calcd for C₁₁H₉O₂N: 187.0633;found: 187.0627.b. Synthesis of compound 202. A mixture of compound 201 (5.01 g, 27.0mmol) in 30 mL dioxane was heated to 75° C., and then a solution of SeO₂in 5:1 dioxane-H₂O (36 mL) was added dropwise. The mixture was heated toreflux with stirring for 4.5 h, and was filtered and the filtrate wasevaporated. The residue was dissolved in chloroform (50 mL) and washedwith water (3×50 mL), dried (MgSO₄) and evaporated. The residue waschromatographed, eluting with CHCl₃, yielding 3.48 g, in 65% yield; mp146.0-147.5° C.; IR (CHCl₃) 1702; ¹H NMR (CDCl₃) δ 6.18 (s, 2H), 7.45(s, 1H), 7.63 (d, 1H, J=4.4), 8.41 (s, 1H), 8.96 (d, 1H, J=4.4), 10.35(s, 1H); ¹³C NMR (CDCl₃) δ 100.4, 102.3, 106.3, 121.4, 124.7, 135.7,148.0, 148.3, 150.8, 151.0, 193.4; HRMS calcd for C₁₁H₇NO₃: 201.0426;found 201.0437.C. Synthesis of compound 203. A mixture of compound 202 (400 mg, 2.0mmol) and 2-iodo-4,5-dimethoxyphenylacetic acid (966 mg, 3.0 mmol,Qandil, A. M., Synthesis, 1999, 2033-2035) in acetic acid (3.5 mL) andTEA (0.31 mL) was heated to reflux with stirring for 90 min. The mixturewas cooled to about 70° C., was poured into water, and the resultingmixture was stirred for 30 min with no additional heating. The entiremixture was then evaporated under vacuum and the residue waschromatographed eluting with 97:3 chloroform-methanol, to provide 725mg, in 73% yield; ¹H NMR (DMSO-d₆) δ 3.47 (s, 3H), 3.72 (s, 3H), 6.24(s, 2H), 6.67 (s, 1H), 6.83 (d, 1H, J=4.7), 7.23 (s, 1H), 7.33 (s, 1H),7.41 (s, 1H), 8.15 (s, 1H), 8.44 (d, 1H, J=4.7); HRMS calcd forC₂₁H₁₆INO₆H: 506.0101; found: 506.0110.d. Synthesis of compound 204. Thionyl chloride (5 mL) was added dropwiseto a mixture of compound 203 (1.51 g, 3.0 mmol)in absolute ethanol (125mL), and the mixture was refluxed for 5 h with stirring. The mixture wascooled and evaporated under vacuum. The residue was dissolved inchloroform (250 mL) and washed with sat. NaHCO₃ (3×250 mL), dried,evaporated, and chromatographed eluting with chloroform, to provide 1.59g, in 99% yield, as an orange oil; ¹H NMR (CDCl₃) δ 1.31 (t, 3H, J=7.0),3.47 (s, 3H), 3.80 (s, 3H), 4.31 (q, 2H, J=7.0), 6.07 (d, 2H), 6.39 (s,1H), 6.71 (d, 1H, J=4.6), 7.18 (s, 1H), 7.29 (s, 2H), 8.20 (s, 1 H),8.38 (d, 1H, J=4.6); ¹³C NMR (CDCl₃) δ 14.3, 55.9, 56.1, 61.8, 88.2,99.6, 102.0, 106.4, 113.5, 119.1, 121.4, 123.6, 132.4, 136.4, 139.2,140.2, 146.8, 147.5, 148.5, 149.3, 150.6, 166.0; HRMS calcd forC₂₃H₂INO₆H: 533.0413; found 533.0419.

Example 2 Synthesis of Compound 206

A mixture of compound 205 (40 mg, 0.1 mmol) in 10% NaOH (45 mL) andethanol (10 mL) was heated to reflux with stirring overnight. Themixture was concentrated to dryness and water (30 mL) was added. Theresulting mixture was acidified by the addition of acetic acid and thefree acid was then isolated by filtration. After complete drying, 32 mg(87%) was obtained. This acid was added to thionyl chloride and themixture was heated to reflux for 4 h. The excess thionyl chloride wasremoved under vacuum, and N,N-dimethylethylenediamine (5 mL) was added,and the mixture was stirred at ambient temperature for 30 minutes. TheN,N-dimethylethylenediamine was evaporated and the residue was dissolvedin chloroform (25 mL) and washed with sat. NaHCO₃ (3×25 mL), andextracted into dilute aqueous HCl (3×25 mL). The combined aqueousextracts were washed with chloroform (2×25 mL), were basified (30%NaOH), and back-extracted into ethyl acetate (3×30 mL). The organicphases were dried (MgSO₄) and evaporated, and the residue waschromatographed eluting with 96:4 chloroform-methanol, providing 18 mg,in 52% yield; ¹H NMR (CDCl₃+1 drop CD₃OD) δ 2.34 (s, 6H), 2.69 (t, 2H,J=6.2), 3.70 (t, 2H, J=6.2), 4.01 (s, 3H), 4.10 (s, 3H), 6.11 (s, 2H),7.35 (s, 1H), 7.75 (s, 1H), 7.79 (s, 1H), 7.93 (s, 1H), 8.29 (s, 1H),9.59 (s, 1H); ¹³C NMR (CDCl₃+1 drop CD₃OD) δ 37.5, 45.2, 55.9, 56.0,58.3, 99.3, 102.0, 102.1, 106.2, 106.3, 117.7, 120.8, 120.9, 123.9,125.7, 129.6, 136.1, 141.6, 144.7, 148.7, 149.6, 149.9, 150.6, 170.0;HRMS calcd for C₂₅H₂₅N₃O₅H: 448.1872; found 448.1865.

Example 3 Synthesis of Compound 208

A solution of compound 207 (246 mg, 0.5 mmol) in acetonitrile (800 mg)was transferred to the photoreactor apparatus and was degassed bynitrogen purge for 30 minutes. The solution was then irradiated througha vycor filter for 90 minutes. The mixture was removed from thephotoreactor, and an equal portion of 207 (246 mg, 0.5 mmol) inacetonitrile (800 mg) was reacted according to the same procedure. Thesolution, which had turned deep red during irradiation, was evaporatedand the residue was chromatographed eluting with 98:2chloroform-methanol, to provide 85 mg of compound 208, in 24% yield; mp231-233° C.; ¹H NMR (CDCl₃) δ 3.13 (m, 2H), 3.55 (m, 3H), 3.96 (s, 3H),4.00 (s, 3H), 6.11 (d, 2H), 6.89 (s, 1H), 7.35 (s, 1H), 7.36 (s, 1H),7.40 (s, 1H), 9.02 (s, 1H); ¹³C NMR (CDCl₃) δ 25.6, 40.2, 56.2, 56.4,64.5, 99.1, 101.9, 106.5, 107.4, 112.2, 124.2, 124.7, 124.9, 129.1,137.6, 143.7, 145.6, 148.5, 149.0, 149.2, 150.1; HRMS calcd forC₂₁H₁₉NO₅H: 366.1341; found: 366.1356.

The intermediate compound 207 was prepared as follows.

a. Synthesis of compound 207. Sodium borohydride (197 mg, 5.2 mmol) wasadded to a mixture of 104 (275 mg, 0.52 mmol) in ethanol (15 mL) at roomtemperature, and the mixture was heated to reflux with stirring for 1 h.The mixture was cooled and quenched by the addition of water (1 mL), andwas then evaporated under vacuum. The residue was partitioned betweenchloroform (50 mL) and sat. NaHCO₃ (50 mL), and the organic phase waswashed with sat. NaHCO₃ (2×50 mL), dried (MgSO₄), and evaporated,yielding 234 mg, in 92% yield; mp 90-92° C.; ¹H NMR (CDCl₃) δ 1.25 (m,1H), 1.40 (m, 1H), 3.18 (m, 1H), 3.50 (m, 1H), 3.65 (m, 1H), 3.85 (s,3H), 3.86 (s, 3H), 6.13 (s, 2H), 6.82 (s, 1H), 6.97 (d, 1H, J=4.6), 7.21(s, 1H), 7.40 (s, 1H), 7.42 (s, 1H), 8.51 (d, 1H, J=4.6); 13C NMR(CDCl₃) δ 35.0, 51.0, 56.1, 56.2, 65.3, 90.4, 99.4, 101.8, 106.4, 110.5,120.7, 122.2, 124.8, 136.1, 144.3, 146.7, 147.5 148.2, 148.7, 149.8,150.4; HRMS calcd for C₂₁H₂₀INO5H: 494.0464; found: 494.0480.

Example 4 Synthesis of Compound 209

Methanesulfonyl chloride (31 μL, 0.4 mmol) was added to a cooledsolution of 208 (65 mg, 0.18 mmol) and TEA (0.1 mL, 0.7 mmol) inmethylene chloride (15 mL). Cooling was removed and the solution wasstirred for 2 h at room temperature. The reaction mixture was thenwashed with sat. NaHCO₃ (3×30 mL), was evaporated, and the residue waschromatographed in chloroform, providing 75 mg of the mesylate, whichwas not characterized, but was used immediately in the next step. Themesylate was added to 15 mL of 95% ethanol, and sodium cyanide (30 mg,0.6 mmol) was added. The mixture was heated to reflux for 7 h. Themixture was cooled and evaporated, and the residue was dissolved inchloroform (50 mL) and washed with water (3×50 mL), was evaporated, andchromatographed in 99:1 chloroform-methanol, provid 46 mg (65%). Thismaterial, which was of about 85% purity, was recrystallized fromethanol/methanol/hexanes, providing 25 mg of material of purity >95%; mp212-214° C.; ¹H NMR (CDCl₃) δ 2.41 (m, 2H), 3.22 (m, 1H), 3.45 (m, 1H),3.53 (m, 1H), 3.99 (s, 3H), 4.02 (s, 3H), 6.15 (s, 2H), 6.90 (s, 1H),7.36 (s, 1H), 7.42 (s, 2H), 9.08 (s, 1H); ¹³C NMR (CDCl₃) δ 22.4, 28.5,35.1, 56.3, 56.4, 98.9, 102.0, 106.7, 107.5, 111.4, 118.3, 124.0, 124.1,124.4, 128.6, 135.4, 143.7, 146.0, 148.9, 149.5, 149.6, 150.4; HRMScalcd for C₂₂H₁₈N₂O₄H: 375.1346; found 375.1332.

Example 5

Other representative compounds of the invention (e.g. compounds 215,216, 218, and 219) can be prepared as illustrated in Schemes D-F.

Example 6

The following illustrate representative pharmaceutical dosage forms,containing a compound of formula I or II (‘Compound X’), for therapeuticor prophylactic use in humans.

(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 01 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Injection 3 (1 mg/ml) mg/ml ‘Compound X’ (free base form) 1.0Citric Acid 0.1% D5W q.s. ad 1 mL

(vii) Aerosol mg/can ‘Compound X’ 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound selected from:

or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 6. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim1 which is:

or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition comprising a compound selected from:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10 whichcomprises the compound:

or a pharmaceutically acceptable salt thereof.
 12. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 13. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 14. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 15. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 16. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 17. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.
 18. The pharmaceuticalcomposition of claim 10 which comprises the compound:

or a pharmaceutically acceptable salt thereof.